The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin.

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.     

Calmodulin antagonist W 13 prevents DNA repair after bleomycin treatment of human urological tumor cells growing on extracellular matrix

1. The combined application of DNA strand-scission agents (bleomycin) and inhibitors of recovery from lethal damage (calmodulin antagonist W-13) could be a novel and potentially important approach to cancer therapy. 2. As determined by alkaline elution, both DNA-DNA and DNA-protein cross-links in bleomycin-treated cells were revealed by the presence of the proteinase K assay. 3. This lethal effect could be potentiated by the addition of calmodulin antagonist W-13 that prevents the repair of DNA strand breaks and DNA cross-links caused by bleomycin. 4. The results indicated that combinations of bleomycin and W-13 were more effective than treatment with either single agent. Isobologram analysis suggests synergistic effect of these drugs. 5. Therefore, the rational use of combinations of DNA-strand-scission agents and inhibitors of recovery from lethal damage based on mechanistic considerations should result with improved therapeutic regimens for the treatment of cancer.     

Cellular responses to the DNA strand-scission enediyne C-1027 can be independent of ATM, ATR, and DNA-PK kinases

The current paradigm based upon ionizing radiation (IR) studies states that cells deficient in either ataxia-telangiectasia-mutated kinase (ATM) or related phosphatidylinositol 3 (PI 3) -kinases (ATR and DNA-PK) are hypersensitive to DNA strand breaks because they are unable to rapidly activate downstream effectors such as p53. Here we have contrasted cell responses to IR and C-1027, a radiomimetic antibiotic that induces DNA strand breaks. At equal levels of DNA double strand breaks, cell lines with inactive ATM or other phosphatidylinositol 3-kinases displayed classical hypersensitivity to IR but not to C-1027. Moreover, phosphorylation of p53 Ser-15 induced by C-1027 was independent of ATM, ATR, or DNA-PK function. We have concluded that the model based on IR studies cannot always be directly applied to DNA damage induced by other strand-scission agents.     

Single-strand nicking of DNA in vitro by neocarzinostatin and its possible relationship to the mechanism of drug action.

Neocarzinostatin, a protein antibiotic with anti-tumor activity was found to place single-strand scissions in DNA in an in vitro reaction. The drug's cutting activity was strongly dependent on the presence of 2-mercaptoethanol or dithiothreitol but some cutting did take place in the absence of reducing agent at very high drug levels and prolonged incubation. The requirement for reducing agents could not be replaced with NAD+, FAD, NADH or H2O2 and the strand-scission reaction was not affected by Mg2+, EDTA or intercalating agents. Similar profiles of heat-inactivation of neocarzinostatin were found whether activity was measured by the scission of DNA strand either in vitro or in HeLa cells treated with the drug. Furthermore, both of these parameters corresponded closely with the ability of the modified drug to inhibit DNA synthesis and growth of HeLa cells. By column isoelectric focusing it was shown that all four activities are associated with the same protein band (pH 3.28). From these data we conclude that the cytotoxic activity of neocarzinostatin and the nicking of DNA strands in vitro appear to reside in the same protein.     

C-1027-induced alterations in Epstein-Barr viral DNA replication in latently infected cultured human Raji cells: relationship to DNA damage.

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.     

Escherichia coli 5S RNA A and B conformers. Characterisation by enzymatic and chemical methods

The structures of the two stable conformers of Escherichia coli 5 S RNA, the and B form, were compared. Information about the structures were obtained using the methods of limited enzymatic hydrolysis and chemical modification of accessible nucleotides. Base-specific modifications were performed for adenosines and cytidines using diethylpyrocarbonate and dimethylsulfate in combination with a strand-scission reaction at the modified site. Base-specific (RNase T1) as well as conformation-specific (nuclease S1, cobra venom nuclease) enzymes were employed for the limited enzymatic hydrolysis. Clear differences in the accessibility of the two 5 S RNA conformers to the enzymes and the chemical reagents were established and the regions with altered reactivities were localized in the 5 S RNA structure. The results are consistent with the disruption of the secondary structural interactions in helix II and partly in helices III and IV during the transition from the A to the B form. (The numbering of the helices is according to the generally accepted Fox and Woese model.) In addition some regions presumably involved in the tertiary structure are distorted. There is evidence, however, for the new formation of structural regions between two distant sites in the 5 S RNA B form. The results enable us to refine the existing 5 S RNA A-form model and provide insight into the structural dynamics that lead to the formation of the 5 S RNA B form.     

DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities.

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.     

Yeast DNA polymerases: antigenic relationship, use of RNA primer and associated exonuclease activity.

Highly purified preparation of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity. The following results were obtained. 1. Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes. 2. DNA polymerase A is capable of using oligo(ribouridylic acid) as a primer for the polymerization of dTMP. This reaction is not catalyzed by polymerase B to any significant extent. 3. Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3' end of polynucleotides. The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule.     

制备炭疽芽胞杆菌检测基因芯片的初步研究

建立制备炭疽芽胞杆菌检测基因芯片的技术,并探讨研制检测炭疽芽胞杆菌基因芯片的方法。酶切炭疽芽胞杆菌的毒素质粒和荚膜质粒,通过建立质粒DNA文库的方法获取探针,并打印在经过氨基化修饰的玻片上,制成用于炭疽芽胞杆菌检测的基因芯片。收集了290个阳性克隆探针,制备了检测炭疽芽胞杆菌的基因芯片。提取炭疽芽胞杆菌质粒DNA与基因芯片杂交,经ScanArray Lite芯片阅读仪扫描得到初步的杂交荧光图像。通过分析探针的杂交信号初步筛选出273个基因片段作为芯片下一步研究的探针。     

Isolation of a non-tumor-inducing mutant of the Ti plasmid of Agrobacterium tumefaciens strain B6.

A nonpathogenic mutant of Agrobacterium tumefaciens strain B6 was isolated and its properties compared with the parental strain in an effort to localize the mutation. Both B6 and its mutant (B6-95) had similar colony color and morphology, were ketolactose positive, utilized octopine, and contained plasmid DNA. Kinetic analysis of DNA reannealing showed that total DNA homology and plasmid DNA homology between B6 and B6-95 was at least 90%. The length of both plasmids was found to be 58 micrometer. Plasmid DNA from both B6 and the mutant was digested with endonucleases and the fragments separated by agarose gel electrophoresis. In all cases the pattern for B6 was identical with that of B6(-95). The Ti plasmid from B6 and the mutant was transferred to an avirulent, plasmidless strain of A. tumefaciens by in vitro conjugation and transformation. All of the B6 transconjugants and transformants were virulent, whereas all of the mutant transconjugants and transformants were avirulent. Electrophoretic patterns of endonuclease-digested plasmid DNA from transformants were identical to those of plasmid DNA from B6. Therefore, we conclude that the virulence mutation lies on the Ti plasmid.     

Effects of dietary polyunsaturated fats and vitamin E on aging and peroxidative damage to DNA

Peroxidative damage to DNA was studied in rats fed either a diet with 10% tocopherol-stripped corn oil and 30 IU DL-alpha-tocopherol acetate/kg (group A), the same diet without vitamin E (group B), a diet with 24% corn oil without vitamin E (group C), or the diet of group A for 10 months and then the diet of group C for 4 months (group D). After 3, 6, 9, and 14 months of feeding the diets, body weights, motoric activities, testicular weights, and lipid-soluble fluorophores in testes were measured. Groups A and B had higher hepatic DNA template activities at 9 and 14 months than group C, and group A had higher testicular DNA template activities than groups B and C at 6, 9, and 14 months. Hepatic DNA template activity of group C decreased from 6 to 9 and from 9 to 14 months. Group C hepatic DNA transcribed less long RNA than that of groups B and D, and more short RNA than groups B and D. Group A testicular DNA transcribed more medium-length RNA than that of groups B and D, and less short RNA than that of groups B, C, and D. DNA-bound tryptophan and DNA crosslinking were inversely related to DNA template activities. DNA damage correlated with other biochemical and physiological changes that are characteristic of cellular impairment in aging and disease.     

Lactic acid bacteria isolated from soy sauce mash in Thailand

Fourteen sphere-shaped and 30 rod-shaped lactic acid bacteria were isolated from soy sauce mash of two factories in Thailand. These strains were separated into two groups, Group A and Group B, by cell shape and DNA-DNA similarity. Group A contained 14 tetrad-forming strains, and these strains were identified as Tetragenococcus halophilus by DNA similarity. Group B contained 30 rod-shaped bacteria, and they were further divided into four Subgroups, B1, B2, B3, and B4, and three ungrouped strains by phenotypic characteristics and DNA similarity. Subgroup B1 contained 16 strains, and these strains were identified as Lactobacillus acidipiscis by DNA similarity. Subgroup B2 included two strains, and the strains were identified as Lactobacillus farciminis by DNA similarity. Subgroup B3 contained five strains. The strains had meso-diaminopimelic acid in the cell wall, and were identified as Lactobacillus pentosus by DNA similarity. The strains tested produced DL-lactic acid from D-glucose. Subgroup B4 contained four strains. The strains had meso-diaminopimelic acid in the cell wall, and they were identified as Lactobacillus plantarum by DNA similarity. Two ungrouped strains were homofermentative, and one was heterofermentative. They showed a low degree of DNA similarity with the type strains tested, and were left unnamed. The distribution of lactic acid bacteria in soy sauce mash in Thailand is discussed.     

Characterisation of rye B chromosome DNA by DNA/DNA hybridisation

The DNAs of wheat and rye plants with rye B chromosomes have been compared with wheat, rye and oats DNAs by DNA/DNA hybridisation. The presence of DNA from B chromosomes made no significant difference to the proportion of repeated sequence DNA. The repeated sequence fractions of these cereal DNAs were quantitatively divided into eight different groups on the basis of the amount of DNA/DNA hybridisation occurring between the different DNAs. Rye A and B chromosomes contained similar proportions of three of the groups. These results, together with estimates of the thermal stabilities of all the renatured DNA duplexes suggest that rye B chromosome DNA is very similar to rye A chromosome DNA in the proportion and heterogeneity of its repeated sequences.     

Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta.     

Functional conservation of multiple elements in yeast chromosomal replicators.

Replicators that control the initiation of DNA replication in the chromosomes of Saccharomyces cerevisiae retain their function when cloned into plasmids, where they are commonly referred to as autonomously replicating sequences (ARSs). Previous studies of the structure of ARS1 in both plasmid and chromosome contexts have shown that it contains one essential DNA element, A, that includes a match to the ARS consensus sequence (ACS), and three additional elements, B1, B2, and B3, that are also important for ARS function. Elements A and B3 are bound by a candidate initiator protein called the origin recognition complex and ARS-binding factor 1, respectively. Although the A and B3 elements have been found in other ARSs, sequence comparisons among ARSs have failed to identify B1- and B2-like elements. To assess the generality of the modular nature of yeast replicators, linker substitution mutagenesis of another yeast chromosomal replicator, ARS307, was performed. Three DNA sequence elements were identified in ARS307, and they were demonstrated to be functionally equivalent to the A, B1, and B2 elements present in ARS1. Despite the lack of DNA sequence similarity, the B1 and B2 elements at each ARS were functionally conserved. Single-base substitutions in the core of the ARS1 B1 and B2 elements identified critical nucleotides required for the function of the B1 element. In contrast, no single-point mutations were found to affect B2 function. The results suggest that multiple DNA sequence elements might be a general and conserved feature of replicator sequences in S. cerevisiae.     

The DNAs of the A and B chromosomes of the mealy bug,Pseudococcus obscurus

When the DNA of mealy bugs carrying B chromosomes (+ B:DNA) was compared to the DNA of individuals not possessing Bs (-B:DNA), no significant differences were found using isopycnic centrifugations in CsCl or thermal denaturation analyses. Both DNAs had buoyant densities of 1.693 g/cm³ in neutral CsCl gradients and 1.748 g/cm³ in alkaline CsCl gradients. Satellite DNAs were not detected. The average T_m of +B:DNA was 67.9° C in 0.1 SSC while -B:DNA had an average T_m of 67.4° C in the same solution. However, in situ molecular hybridizations with complementary RNAs (cRNAs) transcribed in vitro from each type of DNA showed considerable differences with regard to the amount of labeling of B chromosomes. Using cRNA to +B:DNA, the average number of silver grains over a B chromosome was 2.1 × the average number of silver grains over individual non-B chromosomes (A chromosomes). In contrast, the ratio (B/A) using cRNA to -B:DNA was less than 0.14. The results are interpreted as meaning that very little DNA is shared in common by both A and B chromosomes.     

The effect of hydrogen ions on the B-A transition in DNA

The effects of hydrogen ions binding to DNA on its secondary structure and B to A transition were studied by methods of X-ray diffraction and infrared spectroscopy. Helical parameters of DNA molecules with different degrees of protonation were determined. It was shown that H+-ions binding stabilize the B form of DNA in fibers in the wide range of water and inorganic salt content. Only 0.03 H+-ions bound to each nucleotide are sufficient to prevent B to A transition caused by a relative humidity decrease in DNA fibers, containing 4% of NaCl. The effective stabilization of the DNA B form by H+-ions binding is explained by modifications in DNA - solvent molecules interactions, especially in the major groove of double helices.     

Total nucleotide sequences of the infectious cloned DNAs of bean golden mosaic virus

Complete nucleotide sequences of the infectious cloned DNA components (DNA A and B) of bean bolden mosaic virus were determined. The DNA A (2585 nucleotides) and DNA B (2647 nucleotides) have little sequence homology with each other, but both A and B contain a common region of 205 nucleotides. A possible large hairpin structure is detected in the common region. Nucleotide sequences of DNAs A and B revealed the presence of 8 potential coding regions for proteins (m.w. greater than 10,000). Among them, four open reading frames (ORFs 1-4) encode proteins of m.w. 30,000 or greater, and are individually coded in virion DNA A and B senses (+) and their complementary senses (-), respectively. The other four ORFs 5-8 are in virion DNA B(+) and its complementary sense (-). All of the ORFs 1-4 have regulatory signals for RNA synthesis (TATAA/T) in the region 5' upstream from a potential start codon ATG.     

Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination

We have previously purified and characterized wheat germ DNA polymerases A and B. To determine the role played by DNA polymerases A and B in DNA replication, we have measured the level of their activities during wheat embryo germination. The level of cellular proteins known to be associated with DNA synthesis such as PCNA and DNA primase were also investigated. The activity of DNA polymerase A gradually increased reaching a maximal level at 12 h after germination. Three days later, only a residual activity was detected. DNA polymerase B showed the same pattern during germination with very similar changes in activity. Our results indicate a striking correlation between maximal activities of DNA polymerase A, DNA polymerase B and optimal levels of DNA synthesis. These results support a replicative role of these enzymes. The activity of wheat DNA primase that copurifies with DNA polymerase A also increases during wheat germination. Taking together all its properties, and in spite of its behaviour with some inhibitors, DNA polymerase A may be considered as the plant counterpart of animal DNA polymerase . Concerning DNA polymerase B we have previously shown that PCNA stimulates its processivity. Besides studying the changes of DNA polymerases A and B and DNA primase we have also studied changes in PCNA during germination. We show that PCNA is present in wheat embryos at a constant relatively high level during the first 24 h of germination. After 48 h, the absence of PCNA is concomitant with an important decrease in DNA polymerase B activity. In this report we confirm the behaviour of DNA polymerase B as a -like activity.Département de Biologie, Université de Drah-Lmraz,Fez, Maroc